Tert-BUTYLHYDROQUINONE (TBHQ)
First draft prepared by
Ms Elizabeth Vavasour
Toxicological Evaluation Division, Bureau of Chemical Safety
Food Directorate, Health Protection Branch, Health Canada
Ottawa, Ontario, Canada
Explanation
Biological data
Biochemical aspects
Absorption, distribution, and excretion
Biotransformation
Effects on enzymes and other biochemical parameters
Toxicological studies
Long-term toxicity studies
Special studies on lung toxicity
Special studies on carcinogenesis
Special studies on tumour promotion
Special studies on teratogenicity
Special studies on genotoxicity
Special studies on the renal pelvic epithelium
Special studies on potentiation or inhibition of
cancer
Observations in humans
Comments
Evaluation
References
1. EXPLANATION
Tert-Butylhydroquinone (TBHQ) was previously evaluated by the
Committee at the nineteenth, twenty-first, thirtieth and
thirty-seventh meetings (Annex 1, references 38, 44, 73, 74 and 94).
At the thirty-seventh meeting, the previously established temporary
ADI of 0-0.2 per kg of body weight was extended, pending receipt of
the results of ongoing long-term toxicity studies in rodents. This ADI
was derived from a NOEL of 1500 mg/kg of feed (equivalent to 37.5/kg
bw/day) in a 117-week feeding study in dogs on the basis of
haematological abnormalities observed at the next highest dose level
of 5000 mg/kg of feed (Annex 1, reference 39). In addition, the
Committee requested clarification of the results of genotoxicity
assays available at that time, which indicated that TBHQ was
clastogenic in both in vitro and in vivo assays, but was
apparently devoid of activity in bacterial mutagenicity assays. At its
present meeting, the Committee reviewed the results of all available
genotoxicity assays with TBHQ, in addition to some new studies on its
metabolism and disposition, its effects on the renal pelvic
epithelium, and its role in the promotion/inhibition of cancer. The
final results of long-term studies in mice and rats were not available
for review.
This monograph addendum summarizes the information on TBHQ that
has become available since the previous evaluation, including the
information evaluated by the thirty-seventh meeting.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
Oral doses of 4 ml of 0.01%, 0.1% or 1.0% butylated
hydroxyanisole (BHA) were administered to male F344 rats. After
3 h, the concentrations of tert-butylquinone (TBQ) (the oxidation
product of TBHQ) detected by HPLC in the forestomach mucosa were
0.0045, 0.045, and 0.0552 µg TBQ/animal, compared to 1.8, 18.8 and
216.3 µg BHA/animal, respectively (Morimoto et al., 1991).
TBHQ was not detected in homogenates of forestomach mucosa from
male F344 rats which had received 4 ml of 0.01% - 2.0% [14C]BHA.
Forestomach homogenates were therefore treated with sodium dodecyl
sulfate in order to reduce TBQ to TBHQ, in which form it could be more
easily measured. The TBHQ content thus generated in the forestomach
homogenates was proportional to the dose of BHA. The ratios of the
total tissue content of TBHQ to the total amount of covalent binding
of 14C in forestomach were 0.010.03%, at p.o. BHA doses of 0.1-2.0%.
The authors concluded that the covalent binding level was an important
indicator of reactive metabolites of BHA (Morimoto et al., 1992).
2.1.2 Biotransformation
Following the administration i.p. of 400 mg/kg bw BHA or
200 mg/kg bw TBHQ to male Wistar rats, two previously undocumented
metabolites, 3- tert-butyl-5-methylthiohydroquinone (TBHQ-5-SMe) and
3- tert-butyl-6-methyl-thiohydroquinone (TBHQ-6-SMe), were detected
in the urine using GC-MS. The authors suggested that these metabolites
resulted from the metabolic conversion of glutathione conjugates of a
quinone or semiquinone form of TBHQ. In rat liver microsomal
preparations, the formation of two GSH conjugates at the 5- and 6-
positions of TBHQ in the presence of an NADPH-generating system,
molecular oxygen and GSH was confirmed. It appeared that glutathione
S-transferase was not required for the reaction. Also, while
inhibitors of cytochrome P-450 markedly reduced formation of TBHQ-GSH
conjugates, indicating its role in the activation of TBHQ to TBQ,
autooxidation was also shown to play a partial role in this reaction
(Tajima et al., 1991).
Benzylthiol derivatives synthesized from TBQ had higher first
reduction potentials than the parent compound. The authors concluded
that TBQ maintained its potential for the generation of active oxygen
species even after its addition to cellular thiols (Morimoto et al.,
1991).
The tert-butyl semiquinone radical was shown to be formed from
TBHQ in aerobic rat liver microsomes in the presence of NADPH. A
concentration of 500 µM TBHQ and 5µM TBQ produced similar reductions
in SOD-inhibitable cytochrome c which was used as an indication of
excess superoxide anion radical production. The authors concluded that
autooxidation of the semiquinone formed from the quinone was
responsible for superoxide formation and that the hydroquinone entered
the redox cycle via autooxidation. TBQ, but not TBHQ, induced toxic
injury to rat hepatocyte plasma membrane as indicated by LDH release
into the culture medium. The authors speculated that semiquinone-
dependent superoxide formation was responsible for the toxic action
(Bergman et al., 1992).
Incubation of TBHQ with horseradish peroxidase and hydrogen
peroxide resulted in its rapid oxidation to TBQ. TBQ-epoxide was also
produced at concentrations of 2.5 mM and higher of hydrogen peroxide.
The presence of horseradish peroxidase was not a requirement for the
production of TBQ-epoxide from TBQ (Tajima et al., 1992).
Three glutathione conjugates were generated by the incubation of
TBHQ with glutathione; two of these were monoconjugates at the 5 or 6
positions ( tert-butyl group at position 2) and one was a 5,6
diconjugate. The redox potentials for the conjugates were twice those
for the unconjugated hydroquinone. The monoconjugates showed an
approximately 10-fold increase in redox cycling activity (oxygen
consumption in the presence of a reducing agent) compared with TBHQ,
whereas the diconjugate showed a 2-fold increase compared with TBHQ
None of the major glutathione S-transferase isoenzymes were required
for the formation of glutathione conjugates from TBHQ (van Ommen
et al., 1992).
Incubation of TBHQ in phosphate-buffered saline resulted in the
generation of the semiquinone radical through autooxidation,
accompanied by the formation of superoxide anion, hydroxyl radical and
hydrogen peroxide as detected by electron spin resonance (ESR)
spectroscopy. The addition of prostaglandin H synthase resulted in a
substantial increase of semiquinone production with concomitant
production of reactive species. Under the conditions of the assay,
lipoxygenase had no effect on the formation of the semiquinone. The
presence of either prostaglandin H synthase or lipoxygenase was found
to substantially accelerate the metabolism of TBHQ to TBQ compared
with the rates of autooxidation. In an in vivo study, male Wistar
rats were fed diets containing 1.5% BHA for 14 days, with concurrent
administration of prostaglandin H synthase inhibitors acetylsalicylic
acid (0.2%) or indomethacin (0.002%) in the drinking-water. Both
agents produced a significant decrease in the amount of TBQ excreted
into the urine compared with controls receiving drinking-water only,
while the urinary excretion of BHA and its metabolites, TBHQ and TBQ,
was not different between groups (46.9%, 45.4% and 43.5% of the
ingested dose during urine collection in the control, indomethacin and
acetylsalicylic acid groups, respectively). The results suggested an
in vivo role for prostaglandin H synthase in the metabolism of TBHQ
to TBQ (Schilderman et al., 1993a).
2.1.3 Effects on enzymes and other biochemical parameters
Groups of 50 rainbow trouts were fed 0 or 5.6 mmol/kg of diet of
TBHQ (0.1%), BHT, BHA, or ethoxyquin for 6 weeks. The treated trouts
had reduced liver weight/body weight ratios. Compared to controls,
TBHQ-treatment led to a decrease in hepatic microsomal protein and
cytochrome P-450 contents, and nitroanisole-O-demethylase activity. In
contrast, the hepatic activities of benzo-[a]-pyrene hydroxylase,
epoxide hydratase, ethoxycoumarin-O-deethylase, and NADPH-cytochrome
c-reductase were elevated following TBHQ-treatment (Eisele et al.,
1983).
TBHQ was found to be an inducer of quinone reductase in cell
mutants and mouse strains which lack the Ah-receptor. In these target
tissues, agents that induce both phase I and phase II enzymes (such as
polycyclic aromatics) were ineffective (Prochaska, 1987).
TBHQ was found to stimulate the production of superoxide,
hydrogen peroxide, and hydroxyl radicals in microsomes from rat liver
and forestomach. The oxidation product of TBHQ, tert-butylquinone,
exceeded TBHQ in its capacity to induce oxygen radical formation
(Kahl et al., 1989).
TBHQ (30µM) was found to induce quinone reductase activity in
cultures of bone marrow stromal cells from C57BL/6 and DBA/2 mice
(Twerdok & Trush, 1990).
Hepatocytes were isolated from male F344 rats and incubated at
37°C at a concentration of 106 cells/ml with 0.5mM TBHQ. Incubation
with TBHQ resulted in 100% cell death between 1 and 2 h following its
addition to the medium. A drop in intracellular GSH to undetectable
levels was observed in the first hour, prior to the increase in cell
death. Decreases in ATP and reduced protein thiol concentrations were
observed concurrently with the increase in cell death. Although
superoxide anion radicals were generated by autooxidation of TBHQ,
intracellular malondialdehyde was not increased during the incubation
period (Nakagawa & Moldéus, 1992).
2.2 Toxicological studies
2.2.1 Long-term toxicity studies
2.2.1.1 Dogs
Groups of 8 pure bred beagle dogs (4/sex), approximately 6 to 8
months of age were maintained on a commercial diet to which 6%
cottonseed oil was added. TBHQ was added to the test diets at level of
0, 500, 1500 or 5000 mg/kg of feed. Diets were available ad lib for
one hour, 6 days/week. Water was available ad lib at all times. The
dogs were housed individually.
Daily inspection was made for appearance, behaviour, survival and
physical signs. Body weight was determined weekly for the first 12
weeks, and thereafter biweekly. Food intake was determined weekly
during the first 12 weeks, and thereafter periodically. Complete
physical examinations were conducted at various times during the test
period. Haematological and biochemical studies and urine analyses were
made twice before commencement of the feeding study, and at weeks 12,
26, 52, 78 and 104.
Haematological studies consisted of haemoglobin concentration,
haematology, RBC, WBC and differentials. Biochemical studies consisted
of serum BUN, glucose, LDM, SAP, SGPT and bilirubin at week 104 only.
Urinalysis consisted of pH, albumin, glucose, ketone bodies and occult
blood. Because some minor abnormalities were noted in the haematology,
the test period was increased to 117 weeks to permit additional
observations. At week 108, peripheral blood samples were taken. At
week 108, TBHQ was withdrawn from the diet of two dogs of the
high-level group. The animals were maintained in metabolism cages and
24-hour urine samples were collected daily. Blood samples were
collected on days 1, 2, 3, 4, 7, 10 and 13 of this period. An interim
sacrifice of one male and one female of each test group was made at
one year. The remaining animals were sacrificed at week 117. At
autopsy, animals were examined for gross pathological changes. The
liver, kidneys, spleen, heart, brain, lungs, gonads, adrenals, thyroid
and pituitary of all dogs were weighed. Tissues from the following
organs of all dogs of control and high-level groups were examined
microscopically: liver, spleen, gallbladder, stomach, small and large
intestines, pancreas, kidneys, urinary bladder, adrenals, gonads and
adnexa, pituitary, thymus, thyroid, salivary glands, lymph nodes,
heart, lungs, marrow, aorta, skin, muscle, spinal cord and brain. The
liver, stomach, small and large intestines and kidneys of all dogs on
low-and mid-level test diets were also examined microscopically. In
addition, specimens of liver and kidney tissues were prepared for
electron microscopy.
No deaths occurred during the test period. Behaviour and
appearance were normal at all times and physical examinations did not
reveal any treatment-related problems. Growth and food consumption
were similar for control and test groups except at the beginning of
the test when dogs were adjusting to the test diet. Biochemical
studies and urinalysis showed variations within animals and groups,
but none of these differences appeared to be compound-related effects.
Haematologic studies showed variable effects in the high-level group.
RBC were slightly lower in both male and female dogs than their
respective controls. These shifts were also reflected in the
haemoglobin concentration and haematocrits of some animals. At week
99, and a subsequent test period there was a slightly deviation of
reticulocytes in the high-level groups. Peripheral blood smears also
showed more normoblasts as well as occasional increase in erythrocyte
basophilia. These effects were not observed in the lower level groups.
Organ weight and gross pathology and histopathology failed to reveal
any compound related changes. Electron microscopy of liver and kidney
showed normal cellular constituents in test animals. There was no
increase in the endoplasmic reticulum in liver cells of treated
animals (Eastman Chemical Products. 1968).
2.2.2 Special studies on lung toxicity
2.2.2.1 Mice
TBHQ was tested for its potential to produce lung damage in mice
similar to that seen following administration of BHT. Groups of 10
mice were given single intraperitoneal injections of 63, 125, 250, or
500 mg/kg bw TBHQ, or 300, 625, or 1230 mg/kg bw BHT in corn oil.
After 5 days, all animals were necropsied and the lungs were weighed
and examined histomorphologically. TBHQ led to mortality at doses of
125 mg/kg bw (4/10), 250 mg/kg bw (9/10), and 500 mg/kg bw (10/10).
Two of the mice which received 1230 mg/kg bw BHT died before the end
of the observation period. Body weights as well as absolute and
relative organ weights were comparable in all groups. While BHT
produced hyperplasia of pulmonary pneumocytes, TBHQ did not lead to
any treatment-related lung lesions (Krasavage & O'Donoghue, 1984).
2.2.3 Special studies on carcinogenesis
2.2.3.1 Rats
In a study designed to investigate the hyperplastic activity of
BHA and related phenols on rat forestomach, groups of 5-10 male and
female rats were fed powdered diets containing BHA, BHT, TBHQ, or one
of 8 structural analogs for 90 days. TBHQ added to the diet at a
concentration of 2% led to brownish discolorations and mild
hyperplasia of basal cells. The local basal cell hyperplasia did not
tend to differentiate. The hyperplastic activity of TBHQ was however
considerably lower than that of BHA (Altmann et al., 1986).
2.2.3.2 Hamsters
Syrian golden hamsters (16 males/group) were fed a powdered basal
diet containing 0.5% TBHQ (purity &gt 98%) for 20 weeks. This dose is
approximately one quarter of the LD50. The control group received a
basal diet only while 12 other groups received diets containing one of
12 other phenolic compounds in concentrations corresponding to one
quarter of their respective LD50. At the end of the experiment,
animals were killed and organs were fixed. Five sections each were cut
from the anterior and posterior walls of the forestomach, 2 from the
glandular stomach, and 4 from the urinary bladder. Tissues were
processed for histopathology and auto-radiography. Analysis of the
labelling index was made on 4000 cells of urinary bladder epithelium,
3000 cells of pyloric gland epithelium (1000 cells each of fundic
side, middle portion, and pyloric side) and 2000 basal cells of
the forestomach epithelium. Unlike some of the other phenolic
compounds, TBHQ did not induce hyperplasia or tumorous lesions of
the forestomach, the glandular stomach or the urinary bladder.
Furthermore, it did not increase the labelling index in the tissues
investigated (Hirose et al., 1986).
2.2.4 Special studies on tumour promotion
2.2.4.1 Rats
The promoting activity of TBHQ in urinary bladder carcinogenesis
initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in male
F344/Dulrj rats was examined and compared with the effect of
alpha-tocopherol (TP) or propyl gallate (PG). Rats (6-week old) were
treated with 0.05% BBN in drinking-water for 4 weeks. Groups of 20
rats received thereafter control diet or diet containing 2.0% TBHQ,
1.0% PG, or 0.4, 0.75, or 1.5% TP. After 36 weeks, the urinary
bladders of all animals were examined histologically. The incidence of
papillary or nodular hyperplasia was significantly higher in BBN
+TBHQ-treated rats as compared to rats initiated with BBN but
receiving control diet. However, there was no significant differences
for papillomas or cancer. This indicated a weak promoting activity of
TBHQ in BBN-initiated urinary bladder carcinogenesis. TP and PG were
inactive in this respect (Tamano et al., 1987).
The effects of TBHQ and 7 other antioxidants on
7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland,
ear duct, and forestomach carcinogenesis were examined in female
Sprague-Dawley rats. Groups of 20 rats were given a single dose of
25 mg/kg bw of DMBA in 0.5 ml of sesame oil by stomach tubing at 50
days of age. Starting one week later, rats were given a basal diet
containing 0.8% of TBHQ or one of the other antioxidants for 51 weeks.
Controls received basal diet only. Groups of 15 rats served as
carcinogen-free controls and received the different diets without
prior treatment with DMBA. Groups receiving antioxidants had reduced
body weights at the end of the experiment. Histological examinations
revealed a reduced rate of mammary tumour development in TBHQ-treated,
and in DMBA-initiated rats as compared to DMBA-treated controls. The
incidence of ear duct and forestomach tumours was not affected by TBHQ
treatment (Hirose et al., 1988).
The modifying activities of BHA, BHT, and TBHQ and of paired
combinations of these phenolic antioxidants on urinary bladder
carcinogenesis in male F344 rats pretreated with BBN were
investigated. Groups of 20 animals (6-weeks old) were given 0.05% BBN
in their drinking-water for 4 weeks, followed by BHA, BHT, or TBHQ
alone (0.8% each) or in pairs (0.4% each) in their diet for 32 weeks.
Controls received no further treatment after BBN administration. A
decrease in body-weight gain was observed in all antioxidant-treated
groups. The incidence of preneoplastic papillary or nodular
hyperplasia (PN hyperplasia) was slightly but significantly higher in
the group treated with BHA+TBHQ after BBN than in controls receiving
BBN only. The densities of PN hyperplasia were also significantly
increased in all treated groups. However, no synergistic enhancing
effects were observed. No differences were seen with respect to the
incidence and densities of papillomas or carcinomas. Thus BHA, BHT,
and TBHQ all exerted enhancing effects in BBN-induced urinary bladder
carcinogenesis in rats, but no synergism regarding this promotion
occurred (Hagiwara et al., 1989).
The effects of TBHQ on urine composition, bladder epithelial
morphology, and DNA synthesis was studied in comparison with other
antioxidants and bladder tumour promoters. Groups of 10 male Fischer
344 rats, 5-week old, were given powdered basal diet containing 2%
TBHQ or one of 11 other compounds, or basal diet only (controls); two
further groups received two other tumour promoters in their
drinking-water. Five rats in each group were killed after 4 weeks for
estimation of DNA synthesis levels and histopathological examination
by light microscopy. The remaining rats were killed at week 8 for
light and scanning electron microscopic examination of the urinary
bladder. During week 4, fresh urine specimens were obtained from rats
in each group and analyzed for pH as well as electrolyte content. TBHQ
brought about an elevation of DNA synthesis in the urothelium and
produced morphological surface alterations such as the formation of
pleomorphic or short, uniform microvilli and ropey or leafy
microridges. The ability to induce proliferation and cell surface
alterations was common to all bladder tumour promoters investigated.
TBHQ also caused an increase in urinary pH, and a decrease in
potassium and phosphate contents as well as in osmolality (Shibata
et al., 1989).
2.2.5 Special studies on teratogenicity
2.2.5.1 Rats
Groups of 20 female Sprague-Dawley rats were fed basal diet
containing 0, 0.125, 0.25, or 0.50% TBHQ from days 6 to 16 of
gestation. During the mating period and on all other days of
gestation, all treatment groups received control diet only. The
experiment was terminated on day 20 of gestation. Total doses of 970,
1880, or 3600 mg/kg bw/day TBHQ had no effect on the mean body-weight
gain or feed consumption of the dams. The average number of corpora
lutea, implantation sites, viable fetuses, resorptions, fetal body
weights, and mortality did not differ between the control and
treatment groups. A significant number of skeletal variations
(rudimentary ribs) were seen in all groups, but the incidence of these
variations was two times greater in the control group than in any
treatment group. It was concluded that TBHQ was not teratogenic in
rats at all doses employed (Krasavage, 1977).
2.2.6 Special studies on genotoxicity
The results of genotoxicity studies with TBHQ are summarized in
Table 1.
An alkaline elution assay was conducted with DNA extracted from
forestomach epithelium of male F344 rats which had received an oral
dose of 220 mg/kg bw BHA, 0.22 mg/kg bw TBHQ, 0.22 mg/kg bw TBQ, or
0.0022 mg/kg bw TBQ and sacrificed 3 hours later. The BHA or TBHQ
treatments caused no detectable DNA damage, but treatment with TBQ (an
oxidation product of TBHQ) induced dose-related DNA damage. The
elution rate at a dose of TBQ of 0.22 mg/kg bw was significantly
higher than for BHA or controls and was similar to that induced by
15 mg/kg bw MNNG in rat forestomach epithelial cells. The linearity of
the elution pattern suggested that DNA damage was not due to cell
necrosis at a dose of 0.22 mg/kg bw TBQ. TBQ appeared to be cytotoxic
to the forestomach epithelial cells of rats at doses of 220 mg/kg bw
(Morimoto et al., 1991).
The potential of BHA, TBHQ and TBQ to induce oxidative DNA damage
were studied by measuring biological inactivation of single-stranded
bacteriophage phiX-174 DNA and the formation of 7-hydro-8-oxo-2'-
deoxyguanosine (8-oxodG) from dG by these compounds, in vitro, in
the absence and presence of peroxidases. TBHQ, but not BHA or TBQ,
appeared to be a strong inducer of DNA damage as indicated by a strong
inactivation of phage DNA and a potent induction of 8-oxodG formation.
This damage was shown to be due to the formation of superoxide anion,
hydrogen peroxide and hydroxyl radicals. The lack of activity of the
quinone metabolite was attributed by the authors to a lack of
cytochrome P-450 reductase in vitro (Schilderman et al., 1993b).
Table 1. Results of genotoxicity assays on TBHQ
Test System Test Object Concentration of Results Reference
TBHQ
Point mutation
Ames test S. typhimurium &lt 50 µg/ml Negative1 Société Kemin
TA98, TA100, (TA1535) Europa 1982a
TA1535, TA1537, &lt 15 µg/ml
TA1538 (TA1537)
&lt 50 µg/ml
(other strains)
Ames test S. typhimurium &lt 450 µg/plate Negative1 Mueller &
TA98, TA100, (-S9) Lockhart 1983
TA1535, TA1537 &lt 2700 µg/plate
TA1538 (+S9)
Ames test S. typhimurium 1-1000 µg/plate Negative1 Hageman et al.,
TA97, TA100, 1988
TA102, TA104
Ames test S. typhimurium 3-3333 µg/plate Negative2 NTP,
TA97, TA98, unpublished
TA100, TA102 results
Ames test S. typhimurium 0-10 µg/plate Negative1 Matsuoka et al.,
TA97, TA98, (-S9) 1990
TA100, TA102 0.5-250 µg/plate
(+S9)
Table 1 cont'd
Test System Test Object Concentration of Results Reference
TBHQ
Reverse mutation Saccharomyces 100-500 µg/ml Negative1 Rogers et al.,
in yeast cerevisiae D7 (-S9) 1992
50-200 µg/ml
(+S9)
In vitro mammalian Mouse lymphoma &lt 31.3 µg/ml Positive1 Litton Bionetics
point mutation assay cells line L5178Y 1982a
(TK+/-)
In vitro mammalian CHO cells/HGPRT &lt 6 µg/ml (-S9) Negative1 Beilman &
point mutation locus &lt 250 µg/ml Barber 1985
assay (+ S9)
In vitro mammalian Chinese hamster 0.17-3.40 µg/ml Negative3,4 Rogers et al.,
point mutation V79 cells, HGPRT 1992
assay locus
Clastogenic effects and chromosomal aberrations
In vitro chromosomal Chinese hamster V79 &lt 330 µg/ml Positive1 (-S9 Societé Kemin
aberration cells only) Europa 1982b
In vitro chromosomal Chinese hamster 15-62 µmol/l Positive1 (-S9 Phillips et al.,
aberration ovary cells only) 1989
Table 1 cont'd
Test System Test Object Concentration of Results Reference
TBHQ
In vitro Chinese hamster 5-25.2 µg/ml Positive1 NTP,
chromosomal ovary cells (S9) (+S9 only) unpublished
aberration 100.5-300µg/ml results
+S9)
In vitro Chinese hamster 12.5-50 µg/ml Positive1,5 Matsuoka et al.,
chromosomal lung fibroblast (-S9) (+S9 only) 1990
aberration cells 20-40 µg/ml
(+S9)
In vivo chromosomal Mouse bone marrow &lt 200 mg/kg bw Negative Litton Bionetics
aberration assay 1985
In vivo chromosomal Mouse bone marrow 200 mg/kg i.p. Positive Giri et al., 1984
aberration
In vivo chromosomal Mouse bone marrow 2 x 30 mg/kg Positive Giri et al., 1984
aberration bw/day, p.o.
Mouse micronucleus Mouse bone marrow 162, 325, or 650 Negative Litton Bionetics
assay mg/kg bw, p.o. 1982b
Mouse micronucleus Mouse bone marrow 250 mg/kg bw, Positive Société Kemin
assay p.o. Europa 1982c
Table 1 cont'd
Test System Test Object Concentration of Results Reference
TBHQ
Dominant lethal Sprague-Dawley &lt 565 mg/kg Negative Krasavage &
assay rats bw/day, 83 days Farber, 1983
DNA interactions
Mitotic gene S. cerevisiae D7 100-500 µg/ml Negative1 Rogers et al.,
conversion (-S9) 1992
50-200 µg/ml
(+S9)
Sister chromatid Chinese hamster 0.5-16.7 µg/ml Positive1 NTP,
exchange ovary cells (-S9) (+S9 only) unpublished
5-166.7 µg/ml results
(+ S9)
Sister chromatid Chinese hamster 0.17-3.4 µg/ml Negative3,4 Rogers et al.,
exchange V79 cells 1992
Sister chromatid Mouse bone marrow 0.5 - 200 mg/kg Positive Mukerjee et al.,
exchange bw, i.p. 1989
1 Both in the absence and presence of a metabolic activation system derived from rat liver S9 fraction.
2 Both in the absence and presence of a metabolic activation system derived from rat or hamster liver S9
fraction.
3 Cultures with or without added rat or hamster hepatocytes.
4 The highest dose tested did not achieve a 50% reduction in cloning efficiency.
5 The oxidative metabolites of TBHQ, TBQ and TBQ-epoxide induced chromosomal aberrations in this system
in the presence (TBQ and TBQ-epoxide) and absence (TBQ-epoxide) of S-9 metabolic activation
TBHQ induced strand breaks in double stranded phiX-174 relaxed
form I DNA in the presence of micromolar concentrations of copper. The
induced DNA strand breaks were inhibited by a Cu(I) chelator or by
catalase, indicating that a Cu(II)/Cu(I) redox cycle and H2O2
generation were requirements for the observed DNA damage. The authors
concluded that DNA-associated copper in cells may have the potential
to activate phenolic compounds, producing reactive oxygen and
electrophilic phenolic intermediates capable of inducing a spectrum of
DNA lesions (Li & Trush, 1994).
2.2.7 Special studies on the renal pelvic epithelium
This study was performed to investigate early proliferation-
related responses of the renal pelvic epithelium in response to
bladder tumour promoters. Groups of 10 male F344 rats received control
diet or 2% TBHQ. At week 4, the DNA-labelling index of the renal
pelvic epithelial cells was determined from 1000 cells in 5
rats/group. At week 8, kidney sections were prepared for SEM
examination. At the end of the study, body weights of the treated
animals were statistically significantly lower than controls as
demonstrated previously by these investigators (Shibata et al.,
1989; 25% at 4 weeks and 15% at 8 weeks). The mean DNA labelling index
in the renal pelvic epithelium after 4 weeks treatment was 10-fold
higher than in controls, but without statistical significance. Slight
cell surface alterations were observed by SEM after 8 weeks of
treatment in some of the treated rats (Shibata et al., 1991).
2.2.8 Special studies on potentiation or inhibition of cancer
The effects of dietary TBHQ were tested in a multi-organ
carcinogenesis model. Groups of 20 male F344 rats were given a
single intragastric administration of 100 mg/kg bw MNNG, a single
intragastric administration of 750 mg/kg bw EHEN, 2 s.c. injections of
0.5 mg/kg bw MBN and 4 s.c. injections of 40 mg/kg bw DMH. At the same
time, the rats received 0.1% DBN for 4 weeks, followed by 0.1% DHPN
for 2 weeks in the drinking-water for a total carcinogen exposure
period of 6 weeks. Three days after this regime, the rats received in
the diet 1% TBHQ or control diet for 36 weeks. Control groups of 10 or
11 animals received 1% TBHQ alone or basal diet alone. Final body
weights of both TBHQ-treated groups were significantly lower than
those of respective controls (19% for carcinogen-treated control and
9% for basal diet control) and this was reflected in higher relative
liver and kidney weights. Dietary TBHQ following carcinogen treatment
reduced the incidence and multiplicity of colon carcinomas and
slightly reduced the incidence and multiplicity of some preneoplastic
and neoplastic lesions of the kidney. At the same time, this treatment
increased the incidence of oesophageal and forestomach papillomas and
oesophageal papillary or nodular hyperplasia compared with controls
and had no effect on tongue, glandular stomach, duodenum, small
intestine, liver, lung, urinary bladder or thyroid gland (Hirose
et al., 1993).
2.3 Observations in humans
No new information was available.
3. COMMENTS
TBHQ was shown to be oxidatively metabolized to tert-
butylquinone both enzymatically and by autoxidation. The results
of a number of studies indicated that tert-butylquinone
participates in redox cycling through the formation of a semiquinone
radical, such cycling being accompanied by the production of reactive
oxygen species. Glutathione conjugates of tert-butylquinone were
also shown to have the potential to participate in redox cycling
reactions, suggesting that reactive oxygen species might be generated
even after covalent binding of tert-butylquinone to cellular thiols.
The results of bacterial mutagenicity studies available to the
Committee were uniformly negative in both the presence and the absence
of a rat liver metabolic activation system, as observed previously. In
mammalian cell mutagenicity assays, TBHQ was positive in one study at
the thymidine kinase (TK) locus and negative in two studies at the
hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) locus. It was
noted that the TK locus is responsive to reactive oxygen species and
can respond to clastogens, while the HGPRT locus is considerably less
sensitive. Clastogenicity in vitro was demonstrated in four
independent assays and in vivo in one of two independent studies.
Micronuclei were induced in mouse bone marrow in one of two in vivo
studies.
TBHQ caused DNA damage in vitro as a result of the formation of
reactive oxygen species, but did not do so in the rat forestomach
after administration by gavage. tert-Butylquinone, the oxidation
product of TBHQ previously mentioned, did cause DNA damage in this
system at a non-cytotoxic concentration. In this context, it has been
shown that tert-butylquinone is produced in the rat forestomach
following the administration of butylated hydroxyanisole (BHA), TBHQ
being formed as an intermediate.
In a 117-week study, dogs were given TBHQ in their diets. Aside
from a slight decrease in RBC, haemoglobin and haematocrits, more
normoblasts and occasional increases in erythrocyte basophilia seen at
the highest level of 5000 mg/kg of feed, there were no TBHQ-related
effects observed. The NOEL in this study was 1500 mg/kg of feed,
equivalent to 37.5 mg/kg bw/day. This NOEL was the basis of the
temporary ADI established in 1975 (Annex 1, reference 39).
4. EVALUATION
The Committee extended the temporary ADI of 0-0.2 mg/kg bw. The
final results of the long-term studies in mice and rats that are known
to have been completed are required for review in 1997.
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